Angiogenesis
Angiogenesis, the process of new blood vessel formation, is essential for the exponential growth of solid tumors and tumor metastasis. Radiological and cytocidal treatments, combined with regimens involving selective inhibitors of angiogenesis should lead to dramatic reductions in tumor growth. One well-known angiogenesis inhibitor was first discovered as fungal contaminant in bovine endothelial cell cultures that inhibited cell proliferation (Ingber et al. Nature 348:555-557, 1990). The responsible organism was subsequently identified as A. fumagatus, and the product identified as fumagillin, a widely recognized amebicide and antibiotic (McCowen et al., Science 113:202-203 (1951)). Fumagillin was found to be a potent inhibitor of endothelial cell proliferation, but its therapeutic window was insufficient for further clinical advancement. TNP-470, a fumagillin-like derivative with 50-fold higher potency, was subsequently developed from a directed chemical approach (Ingber et al., Nature 348:555-557 (1990), Kusaka et al., Biochem. Biophys. Res. Commun. 174:1070-1076 (1991)). This compound's therapeutic use is limited, however, by its lack of oral availability and dose-limiting neurotoxicity.
Until recently, the molecular target for fumagillin or TNP-470 was unknown. In 1997, the target protein was isolated, purified, and identified by mass spectrometry as the type-2 methionine aminopeptidase (MetAP-2). Fumagillin, and its analogs TNP-470 and ovalicin, are now known to irreversibly bind and potently inhibit MetAP-2, but not the type-1 enzyme (Griffith, E. C., Su, Z., Turk, B. E., Chen, S., Chang, Y-.H., Wu, Z., Biemann, K., and Liu, J. O. (1997) Chem. Biol. 4, 461-471; Sin, N., Meng, L., Wang, M. Q. W., Wen, J. J., Bornmann, W. G., and Crews, C. M. (1997) Proc. Natl. Acad. Sci. USA 94, 6099-6103). Together, these results identified MetAP-2 as a target for the development of anti-angiogenesis agents.
TNP-470 is currently being studied in clinical trials as a therapy to treat cancer. TNP-470 has been shown to mediate its effects in animal models by inhibiting new blood vessel formation at the site of tumor growth (Kruger E. A., and Figg W. D. (2000) Expert Opin. Investig. Drugs 9, 1383-96). How this class of compounds suppresses de novo angiogenesis through inhibition of MetAP-2 remains unclear. Although MetAP-2 has been shown to be a target of these suicide inhibitors, other methionine aminopeptidase enzyme homologs may exist that could contribute to the efficacy or neurological side effects observed in the preclinical animal models and the clinical trials for TNP-470 (Castronovo, V., and Belotti, D. (1996) Eur. J. Cancer 32, 2520-2527).
Methionine Aminopeptidases
Most proteins are encoded by messenger RNAs that dictate that the amino terminus of a nascent polypeptide chain is methionine. More than 60% of all cytosolic proteins lose this initiator methionine, however, in a co-translational excision process catalyzed by methionine aminopeptidase enzymes (Arfin, S. M., Kendall, R. L., Hall, L., Weaver, L. H., Stewart, A. E., Matthews, B. W., and Bradshaw, R. A. (1995) Proc. Natl. Acad. Sci. USA 92, 7714-7718). Methionine aminopeptidases were first isolated from eubacteria and shown to be cobalt-containing enzymes with molecular masses of about 30 kDa (Ben-Bassat et al., J. Bacteriol. 169:751-757 (1987), Suh et al., Gene 169:17-23 (1996), and Miller et al., Proc. Natl. Acad. Sci. (U.S.A.) 91:2473-2477, 1987). Their structures include a novel protease fold, with pseudosymmetry around a pair of cobalt ions (Roderick and Matthews, Biochemistry 32:3907-3912, 1993).
Enzymes with the same substrate specificity, but with larger molecular masses, were isolated from yeast and pig. Highly homologous regions at the C-terminal domain (˜30 kDa) of the eukaryotic and the prokaryotic forms were discovered, although the N-terminal domain of the eukaryotic enzymes was found to be unique (Kendall and Bradshaw, J. Biol. Chem. 267:20667-20673 (1992)). The N-terminal domain of the yeast enzyme contained sequences consistent with two zinc-finger structures, indicating a potential site of nucleic acid interaction. This class of enzyme was designated methionine aminopeptidase type I (MetAP-1). The porcine enzyme lacked the zinc-binding domains, but contained a block of polylysine and aspartic residues within the N-terminal domain, and was described as Type II (MetAP-2). Both isozymes have been found from Archebacteria to man, indicating a critical metabolic function (Arfin et al. Proc. Natl. Acad. Sci. (U.S.A.) 92:7714-7718 (1995), Bradshaw et al., TIBS 23: 263-267 (1998)).
The single prokaryotic and two eukaryotic methionine aminopeptidases that have been characterized extensively demonstrate a substrate preference for proteins that contain a small or uncharged amino acid (Ala, Cys, Gly, Pro, Ser, Thr, or Val) at the second position next to the initiator methionine. In prokaryotes the initiator methionine is formylated while still bound to the initiator tRNA. Removal of the formyl group from tRNAfMet by a peptide deformylase is an absolute prerequisite for methionine removal by MetAPs (Solbiati, J., Chapman-Smith, A., Miller, J. L., Miller, C. G., Cronan, J. E. (1999) J. Mol. Biol. 290, 607-614).
The biological consequence of amino terminal methionine removal is poorly understood, due in part to the essential nature of this process, as revealed by the lethality of bacterial and yeast MetAP deletion mutants (Chang, S. Y., McGary, E. C., and Chang, S. (1989) J. Bacteriol. 171, 4071-4072; Li, X and Chang Y-.H. (1995) Proc. Natl. Acad. Sci. USA 92, 12357-12361). The absolute requirement for methionine removal could be for any of a variety of reasons, including 1) exposure of a glycine residue for myristoylation, 2) destabilization of a protein by targeting its degradation and/or 3) optimal protein function due to stabilization by amino terminal acetylation.
Methionine aminopeptidase-2 is bi-functional. One action is the removal of the N-terminal methionine residues from their protein substrates. MetAP-2 can also bind to and prevent phosphorylation of the α-subunit of the peptide change initiation factor eIF-2 by one or more eIF-2 kinases (Datta et al., Proc. Natl. Acad. Sci. USA 85: 3324-2238 1(1988), Wu et al., J. Biol. Chem. 268:10796-10781 (1993)). This action promotes protein synthesis within the cell. The eIF-2 phosphorylation inhibitory activity of MetAP-2 is unaffected by TNP-470 binding, indicating that the loss of aminopeptidase activity is involved in the anti-angiogenic activity of TNP-470 (Griffith et al., Chem. Biol. 4:461-471 (1997)). The function of methionine aminopeptidase activity in endothelial cell proliferation during tumorigenesis is unclear, although inhibition of MetAP-2 may play a role in altering the stability of one or more protein(s) whose abnormal presence or absence results in endothelial cell dysregulation. Several signaling proteins also appear to be modified by the covalent attachment of myristic acid to a glycine residue which occurs only after the initial amino-terminal methionine removal by MetAP-2 (Peseckis et al., J. Biol. Chem. 267:5107-5114 (1993)). Inhibition of methionine aminopeptidase activity may prevent this covalent attachment, resulting in improper functioning of a signal component specific to endothelial cell cycle regulation (Sin et al. Proc. Natl. Acad. Sci. (U.S.A.) 94:6099-6103 (1997)).
The Use of Methionine Aminopeptidases and Inhibitors of Methionine Aminopeptidases
N-terminal processing agents, such as the methionine aminopeptidases, also function to initiate post-translational peptide or protein modifications which may control or induce activation, translocation, or protein turnover (Bradshaw et al., TIBS 23: 263-267 (1998)). Because this initial processing is important for normal protein functioning, it is possible that alteration of methionine aminopeptidase activity is a factor in a variety of diseases, including angiogenesis. Therapies could then be developed which can modify methionine aminopeptidase activity to restore proper protein processing.
Methionine aminopeptidase activity can also be used to modify recombinant proteins expressed and harvested from E. coli or other expression systems. Recombinant proteins that retain the N-terminal methionine, in some cases, have biological characteristics that differ from the native species that retain the N-terminal methionine, including the induction of undesireable antibodies. Methionine aminopeptidases could lower the cost of manufacturing processes designed produce recombinant proteins that mimic the structure of native species which are used to treat or reduce the symptoms of various diseases (Sandman et al., Biotechnology 13:504-6 (1995)).
Clearly, an understanding of methionine aminopeptidase activity and its role in various tissues can provide useful therapeutic and diagnostic insight into angiogenesis and tumor metastasis. The known MetAP-2 inhibitors are not good candidates for clinical use as angiogenesis inhibitors due to their neurotoxic effects. Differential expression of mammalian MetAP-1, MetAP-2, or other unidentified methionine aminopeptidases may partially or totally account for the observed variation in sensitivity of different cell types to inhibition by TNP-470 and other MetAP-2 inhibitors, and thus account for the toxicity of these compounds.